發(fā)布時(shí)間：2018-12-26 19:39

【摘要】：花生是人類(lèi)最優(yōu)質(zhì)的植物蛋白來(lái)源之一,也是八大類(lèi)容易引起過(guò)敏的食物之一。近年來(lái),由于花生過(guò)敏患者數(shù)量呈明顯上升的趨勢(shì),探索降低花生過(guò)敏性的研究受到了人們的高度重視。Ara h 6是花生主要過(guò)敏原之一,可引發(fā)63%的花生過(guò)敏患者發(fā)生過(guò)敏反應(yīng)。因此,本論文工作以Ara h 6為對(duì)象,研究加工對(duì)花生過(guò)敏原結(jié)構(gòu)及抗原性的影響,旨在闡明Ara h 6經(jīng)加工后的結(jié)構(gòu)變化與其抗原性的關(guān)系。 論文開(kāi)展的研究工作包括花生過(guò)敏原Ara h 6的純化與提取、兔抗Ara h 6多克隆抗體的制備、熱加工和輻照及超高壓微射流對(duì)花生過(guò)敏原Ara h 6結(jié)構(gòu)與抗原性的影響。研究的主要方法、結(jié)果及結(jié)論如下。 1.采用陰離子交換層析法從新鮮花生仁中分離純化花生過(guò)敏原Ara h 6,并通過(guò)SDS-PAGE電泳、MALDI-TOF/MS質(zhì)譜對(duì)分離所得目標(biāo)蛋白進(jìn)行鑒定。結(jié)果表明,該方法分離所得的Arah 6的純度大于95%,得率為18.3%。該方法簡(jiǎn)單、易操作、重現(xiàn)性好,適用于實(shí)驗(yàn)室少量制備。 2.以純化的Ara h 6為抗原,免疫新西蘭大白兔,獲得抗Ara h 6的兔多克隆抗體,通過(guò)間接ELISA和免疫印跡方法鑒定其效價(jià)和特異性,結(jié)果表明,該抗體效價(jià)為1：100,000,特異性強(qiáng),可滿足抗原性評(píng)估的要求。 3.對(duì)Ara h 6純化蛋白和花生粗蛋白分別進(jìn)行不同的熱處理,通過(guò)SDS-PAGE電泳、圓二色譜、紫外光譜、熒光探針光譜檢測(cè)Ara h 6熱加工后的結(jié)構(gòu)變化,并采用間接ELISA方法測(cè)定熱加工后Ara h 6純化蛋白和花生粗蛋白的抗原性變化。結(jié)果表明,Ara h 6經(jīng)熱處理后,其最大紫外吸光值增大,疏水性增強(qiáng),各類(lèi)型二級(jí)結(jié)構(gòu)相互轉(zhuǎn)變,且115℃熱處理可使Ara h 6形成多聚體；抗原性分析結(jié)果則表明,隨著加熱程度的增強(qiáng),Ara h 6純化蛋白和粗蛋白的抗原性降低。由此推斷,Ara h 6蛋白經(jīng)熱加工后的構(gòu)象變化導(dǎo)致了它的抗原性降低,蛋白構(gòu)象對(duì)于Ara h 6蛋白的抗原性起著關(guān)鍵的作用。 4.對(duì)Ara h 6純化蛋白和花生粗蛋白分別進(jìn)行輻照和超高壓微射流處理,通過(guò)SDS-PAGE電泳、圓二色譜、紫外光譜、熒光探針光譜檢測(cè)Ara h 6加工后的結(jié)構(gòu)變化,并采用間接ELISA方法測(cè)定加工后Ara h 6純化蛋白和花生粗蛋白的抗原性變化。結(jié)果表明,Ara h 6經(jīng)輻照或超高壓微射流處理后,蛋白質(zhì)分子展開(kāi),疏水基團(tuán)暴露,二級(jí)結(jié)構(gòu)改變,且輻照處理可使Ara h 6形成多聚體；間接ELISA檢測(cè)結(jié)果表明,隨著輻照劑量的升高或超高壓微射流處理壓力的增加,Ara h 6純化蛋白和粗蛋白的抗原性降低。由此推斷,輻照和超高壓微射流處理破壞了Ara h 6蛋白的結(jié)構(gòu)特征,導(dǎo)致該蛋白抗原性降低,該蛋白的結(jié)構(gòu)穩(wěn)定性對(duì)其抗原性具有至關(guān)重要的作用。
[Abstract]:Peanut is one of the best source of plant protein, and it is one of the eight kinds of food susceptible to allergies. In recent years, because the number of peanut allergy patients is increasing obviously, the research of reducing peanut allergy has been paid great attention to by people,. Ara h 6 is one of the main allergens in peanut. It can cause 63% allergic reaction in peanut allergy patients. Therefore, the effect of processing on the structure and antigenicity of peanut allergen (Ara h 6) was studied in this paper, in order to clarify the relationship between the structure change of Ara h 6 and its antigenicity. The research work in this paper includes the purification and extraction of peanut allergen Ara h6, the preparation of rabbit anti-Ara h6 polyclonal antibody, the effects of thermal processing and irradiation and ultrahigh pressure microjet on the structure and antigenicity of peanut allergen Ara h 6. The main methods, results and conclusions are as follows. 1. The peanut allergen Ara h 6 was purified by anion exchange chromatography from fresh peanut kernel. The target protein was identified by SDS-PAGE electrophoresis and MALDI-TOF/MS mass spectrometry. The results show that the purity of Arah 6 obtained by this method is greater than 95 and the yield is 18.3. The method is simple, easy to operate and reproducible. It is suitable for laboratory preparation. 2. Rabbit polyclonal antibody against Ara h6 was obtained by immunizing New Zealand white rabbits with purified Ara h6 antigen. The titer and specificity of the polyclonal antibody against Ara h6 were identified by indirect ELISA and Western blotting. The results showed that the titer of the antibody was 1: 100000, and the specificity was strong. It can meet the requirements of antigenicity evaluation. 3. The purified protein of Ara H6 and the crude protein of peanut were heat-treated respectively. The structure changes of Ara h 6 after hot processing were detected by SDS-PAGE electrophoresis, circular dichroism, UV spectrum and fluorescence probe spectrum. The antigenicity of Ara h 6 purified protein and peanut crude protein was determined by indirect ELISA method. The results showed that after heat treatment, the maximum UV absorptivity and hydrophobicity of, Ara h 6 increased, and the secondary structures of Ara 6 changed with each other, and the Ara h 6 was formed by heat treatment at 115 鈩，

本文編號(hào)：2392588